HIV Integration Targeting: A Pathway Involving Transportin-3 and the Nuclear Pore Protein RanBP2

Abstract

Genome-wide siRNA screens have identified host cell factors important for efficient HIV infection, among which are nuclear pore proteins such as RanBP2/Nup358 and the karyopherin Transportin-3/TNPO3. Analysis of the roles of these proteins in the HIV replication cycle suggested that correct trafficking through the pore may facilitate the subsequent integration step. Here we present data for coupling between these steps by demonstrating that depletion of Transportin-3 or RanBP2 altered the terminal step in early HIV replication, the selection of chromosomal sites for integration. We found that depletion of Transportin-3 and RanBP2 altered integration targeting for HIV. These knockdowns reduced HIV integration frequency in gene-dense regions and near gene-associated features, a pattern that differed from that reported for depletion of the HIV integrase binding cofactor Psip1/Ledgf/p75. MLV integration was not affected by the Transportin-3 knockdown. Using siRNA knockdowns and integration targeting analysis, we also implicated several additional nuclear proteins in proper target site selection. To map viral determinants of integration targeting, we analyzed a chimeric HIV derivative containing MLV gag, and found that the gag replacement phenocopied the Transportin-3 and RanBP2 knockdowns. Thus, our data support a model in which Gag-dependent engagement of the proper transport and nuclear pore machinery mediate trafficking of HIV complexes to sites of integration.

Interaction of the HIV-1 Intasome with Transportin 3 Protein (TNPO3 or TRN-SR2)

“…If this model gains further support, one could consider a possibility where CA is displaced from PICs by the direct binding of TNPO3 to the intasome rather than CA. Recent reports have also shown that TNPO3 knockdowns reduced HIV-1 integration frequency into gene-dense regions (14). Our findings suggest that TNPO3 could directly associate with the functional nucleoprotein complex and could thus potentially affect the integration site targeting.…”

“…Several candidates have been proposed including viral determinants such as IN (5), CA (6), matrix (7), and viral protein R (8) as well as the following cellular factors: importin-␣ (8), importin-␤ (9, 10), importin 7 (11), TNPO3 (12)(13)(14), Nup153, and Nup358 (10,(13)(14)(15)(16)(17).…”

Single-Cell Imaging of HIV-1 Provirus (SCIP)

“…S6A). (P ut vs. 100nM AZT = 9.555e-05, P ut vs. 1uM = 4.485e-07, P ut vs. 10uM = 4.384e-08, P ut vs. 2uM nev = 0.0003, P ut vs. 10uM nev = 8.572e-13, P ut vs. 100nM ral = 3.466e-11, P ut vs. 500nM ral < 2.2e-16, P ut vs. 1uM ral < 2.2e-16, P ut vs. 100nM CX = 0.0004, P ut vs. 1uM CX = 3.046e-13, P ut vs. 10uM CX = 6.122e-10, KS test Transportin-SR2 (TRN-SR2, TNPO3) is a cellular import factor of SR-rich proteins that is involved in HIV-1 entry in the nuclei of infected cells (24)(25)(26)(27)(28)(29)(30)(31). In fact, after TRN-SR2 knockdown HIV-1 nuclear entry is inhibited and, consequently, the amount of integrated DNA is reduced.…”

RANBP2 (RAN binding protein 2)

“…In summary, RANBP2 aids nuclear import by at least two mechanisms: i) by "capturing" transport receptors through the FG-repeats, it conveys them towards the NPC and reduces the effective concentration of import receptors required for efficient transport, while ii) by interacting with selected cargos in a receptor-independent manner, through the RANBP2 N-ter domain, it increases the overall efficiency of nuclear import. Interestingly, RANBP2 is also implicated in the nuclear delivery and integration of certain human viruses, including Herpes simplex (Copeland et al, 2009) and immunodeficiency virus-1 (HIV-1) (Zhang et al, 2010;Ocwieja et al, 2011;). …”